DNA-Based Enzyme Reactors and Systems

This is an elec onic ep in o he o iginal a icle. This ep in may di e om he o iginal in pagina ion and ypog aphic de ail. Au ho (s): Ti le: Yea : Ve sion: Please ci e he o iginal e sion: All ma e ial supplied ia JYX is p o ec ed by copy igh and o he in ellec ual p ope y igh s, and duplica ion o sale o all o pa o any o he eposi o y collec ions is no pe mi ed, excep ha ma e ial may be duplica ed by you o you esea ch use o educa ional pu poses in elec onic o p in o m. You mus ob ain pe mission o any o he use. Elec onic o p in copies may no be o e ed, whe he o sale o o he wise o anyone who is no an au ho ised use . DNA-Based Enzyme Reac o s and Sys ems Linko, Veikko; Nummelin, Sami; Aa nos, Lau a; Tapio, Kos i; Toppa i, Jussi; Kos iainen, Mau i A. Linko, V., Nummelin, S., Aa nos, L., Tapio, K., Toppa i, J., & Kos iainen, M. A. (2016). DNA-Based Enzyme Reac o s and Sys ems. Nanoma e ials, 6(8), A icle 139. h ps://doi.o g/10.3390/nano6080139 2016 nanoma e ials Re iew DNA-Based Enzyme Reac o s and Sys ems Veikko Linko 1,*, Sami Nummelin 1, Lau a Aa nos 1, Kos i Tapio 2, J. Jussi Toppa i 2 and Mau i A. Kos iainen 1,* 1Biohyb id Ma e ials, Depa men o Bio echnology and Chemical Technology, Aal o Uni e si y, P.O. Box 16100, Aal o 00076, Finland; [email p o ec ed] (S.N.); [email p o ec ed] (L.A.) 2 Depa men o Physics, Uni e si y o Jy askyla, Nanoscience Cen e , P.O. Box 35, Jy äskylä 40014, Finland; [email p o ec ed] (K.T.); [email p o ec ed] (J.J.T.) *Co espondence: [email p o ec ed] (V.L.); [email p o ec ed] (M.A.K.); Tel.: +358-45-673-9997 (V.L.); +358-50-362-7070 (M.A.K.) Academic Edi o : Leonid Gu e ich Recei ed: 8 June 2016; Accep ed: 19 July 2016; Published: 27 July 2016 Abs ac : Du ing ecen yea s, he possibili y o c ea e cus om biocompa ible nanoshapes using DNA as a building ma e ial has apidly eme ged. Fu he , hese a ionally designed DNA s uc u es could be exploi ed in posi ioning pi o al molecules, such as enzymes, wi h nanome e -le el p ecision. This ea u e could be used in he ab ica ion o a i icial biochemical machine y ha is able o mimic he complex eac ions ound in li ing cells. Cu en ly, DNA-enzyme hyb ids can be used o con ol (mul i-enzyme) cascade eac ions and o egula e he enzyme unc ions and he eac ion pa hways. Mo eo e , sophis ica ed DNA s uc u es can be u ilized in encapsula ing ac i e enzymes and deli e ing he molecula ca go in o cells. In his e iew, we ocus on he la es enzyme sys ems based on no el DNA nanos uc u es: enzyme eac o s, egula o y de ices and ca ie s ha can ind uses in a ious bio echnological and nanomedical applica ions. Keywo ds: DNA nano echnology; DNA o igami; sel -assembly; enzyme; cascade eac ions; DNA nanode ice; DNA senso s; d ug-deli e y; nanomedicine 1. In oduc ion In o de o main ain complex me abolic pa hways, na u e uses compa men aliza ion and spa ial o ganiza ion o me abolically ac i e uni s o sepa a e specialized unc ions, con ol ac i i y and gain speci ici y. In he cell, speci ic o ganelles con ol he loca ion and c owding o enzymes, which has a p o ound e ec on hei spa ial ac ion, and ul ima ely allows di e en me abolic pa hways o ope a e a he same ime in close p oximi y, bu in di e en compa men s. Fo example, elec on anspo and oxida i e phospho yla ion a e handled by he mi ochond ion, whe eas a he same ime glycolysis and a y acid biosyn hesis ake place in he cy osol. Fu he mo e, mul iple enzymes esponsible o he indi idual eac ion s eps in a me abolic pa hway a e o en combined in o a single mul i unc ional enzyme o complex in o de o enhance and con ol he eac ion cascade o cycle. Fa y acid syn hase is one o he p ime examples: in animals i combines wo iden ical p o ein chains ha con ain se en di e en ca aly ic ac i i ies equi ed o he biosyn hesis o a y acids. Al hough highly desi able, he p og amming o chosen eac ion cascades and c ea ing a i icial sys ems ha can posi ion and con ine di e en enzymes is s ill a om he complexi y achie ed by na u e. Con olling chemical eac ions by using sel -assembled nanoscale eac o s has consequen ly eme ged as an ac i e a ea o esea ch [1]. Simple compa men aliza ion o enzymes has al eady been achie ed by using di e en nanoscale eac o s. Examples o such sys ems include sol-gel ma e ials [ 2 ], polyme somes [ 3 ], p o ein cages [ 4 – 6 ] and c ys alline s uc u es [ 7 – 9 ]. Po ous polyme somes we e used as nano eac o s o ancho h ee di e en enzymes in o sepa a e loca ions: he lumen, bilaye memb ane and su ace. P o ein cages, such as i us-like pa icles, ha e been u ilized o pack di e en Nanoma e ials 2016,6, 139; doi:10.3390/nano6080139 www.mdpi.com/jou nal/nanoma e ials Nanoma e ials 2016,6, 139 2 o 16 enzymes ha pe o m a coupled cascade eac ion densely inside a po ous p o ein shell. Finally, o example, me al-o ganic amewo ks ha e been designed o ap and encapsula e enzymes and shown o p e en hei agg ega ion and dena u a ion. All o he abo e-men ioned sys ems a e p ime examples on how he posi ioning, sepa a ion and clus e ing o enzymes can be con olled. In his e iew, we ocus on complexes and sys ems ha in ol e unc ional enzymes and no el DNA nanos uc u es (an example o such a sys em is depic ed in Figu e 1). These sophis ica ed DNA nanos uc u es can be p og ammed o o m p ecise and con ollable a angemen s o enzymes a he nanoscale, and hese sys ems a e pa icula ly engaging o a ious applica ions in bioenginee ing and nanomedicine. We ha e di ided his e iew in o ou main sec ions. Fi s , we b ie ly summa ize he de elopmen in s uc u al DNA nano echnology and discuss how DNA mo i s can be combined wi h unc ional enzymes (Sec ion 2). Sec ion 3is de o ed o (s a ic) enzyma ic nano eac o s, and Sec ion 4 co e s he enzyma ic egula o y de ices wi h mechanical unc ion. Finally, in Sec ion 5, con aine s and ca ie s o p o ec ing and deli e ing enzymes a e discussed. Nanoma e ials 2016, 6, 139 2 o 16 di e en enzymes ha pe o m a coupled cascade eac ion densely inside a po ous p o ein shell. Finally, o example, me al-o ganic amewo ks ha e been designed o ap and encapsula e enzymes and shown o p e en hei agg ega ion and dena u a ion. All o he abo e-men ioned sys ems a e p ime examples on how he posi ioning, sepa a ion and clus e ing o enzymes can be con olled. In his e iew, we ocus on complexes and sys ems ha in ol e unc ional enzymes and no el DNA nanos uc u es (an example o such a sys em is depic ed in Figu e 1). These sophis ica ed DNA nanos uc u es can be p og ammed o o m p ecise and con ollable a angemen s o enzymes a he nanoscale, and hese sys ems a e pa icula ly engaging o a ious applica ions in bioenginee ing and nanomedicine. We ha e di ided his e iew in o ou main sec ions. Fi s , we b ie ly summa ize he de elopmen in s uc u al DNA nano echnology and discuss how DNA mo i s can be combined wi h unc ional enzymes (Sec ion 2). Sec ion 3 is de o ed o (s a ic) enzyma ic nano eac o s, and Sec ion 4 co e s he enzyma ic egula o y de ices wi h mechanical unc ion. Finally, in Sec ion 5, con aine s and ca ie s o p o ec ing and deli e ing enzymes a e discussed. Figu e 1. A schema ic iew o an enzyma ic nano eac o buil om DNA ([S] = subs a e, [P] = p oduc ). By aking ad an age o he high add essabili y and modula i y o he DNA nanos uc u es, enzymes can be a ached and a anged wi h nanome e -scale p ecision. As an example, glucose oxidase (GOx, pu ple)–ho se adish pe oxidase (HRP, g een) cascade pai s ha e been assembled in o a con ined eac ion space p o ided by wo ubula DNA o igami nanos uc u es (o ange and yellow cages). 2. Building wi h DNA Molecules and Enzymes 2.1. DNA Nanos uc u es Nad ian ‘Ned’ Seeman pos ula ed a ound 30 yea s ago ha deoxy ibose (DNA) molecules could be used as building ma e ial in c ea ing complex p edesigned nanos uc u es h ough molecula sel - assembly [10]; sequence-complemen a y pa s o single-s anded DNA (ssDNA) molecules can be hyb idized in o double-s anded DNA (dsDNA) domains ( ia Wa son-C ick base-pai ing) and he e o e in o la ge p og ammed shapes. Since hen, s uc u al DNA nano echnology has enjoyed a apid p og ess; nume ous complex nanos uc u es and di e en ab ica ion echniques ha e been in oduced [11] (Figu e 2). A g ea deal o he i s compelling DNA assemblies we e based on ile- like s uc u es ha enabled ab ica ion o wo-dimensional (2D) [12] and h ee-dimensional (3D) c ys als [13], bu ne e heless, he huge up u n in he ield was he in en ion o he ‘DNA o igami’ echnique [14] (Figu e 2a). The o igami app oach is based on olding a long single-s anded DNA sca old s and in o a desi ed shape wi h he help o a se o sho oligonucleo ides (s aples), and i has now become a widely accessible and exploi ed me hod o ab ica e cus om, modula and spa ially- well-de ined 2D [14] and 3D nanos uc u es wi h complex cu a u es, bends and wis s [15–18] (see Figu e 2b,c). La e on, me hods based on sca old- ee ab ica ion [19] (Figu e 2d), polyhed al ende ing [20,21] (Figu e 2e) and shape-complemen a i y [22] (Figu e 2 ) we e in oduced. Figu e 1. A schema ic iew o an enzyma ic nano eac o buil om DNA ([S] = subs a e, [P] = p oduc ). By aking ad an age o he high add essabili y and modula i y o he DNA nanos uc u es, enzymes can be a ached and a anged wi h nanome e -scale p ecision. As an example, glucose oxidase (GOx, pu ple)–ho se adish pe oxidase (HRP, g een) cascade pai s ha e been assembled in o a con ined eac ion space p o ided by wo ubula DNA o igami nanos uc u es (o ange and yellow cages). 2. Building wi h DNA Molecules and Enzymes 2.1. DNA Nanos uc u es Nad ian ‘Ned’ Seeman pos ula ed a ound 30 yea s ago ha deoxy ibose (DNA) molecules could be used as building ma e ial in c ea ing complex p edesigned nanos uc u es h ough molecula sel -assembly [ 10 ]; sequence-complemen a y pa s o single-s anded DNA (ssDNA) molecules can be hyb idized in o double-s anded DNA (dsDNA) domains ( ia Wa son-C ick base-pai ing) and he e o e in o la ge p og ammed shapes. Since hen, s uc u al DNA nano echnology has enjoyed a apid p og ess; nume ous complex nanos uc u es and di e en ab ica ion echniques ha e been in oduced [ 11 ] (Figu e 2). A g ea deal o he i s compelling DNA assemblies we e based on ile-like s uc u es ha enabled ab ica ion o wo-dimensional (2D) [ 12 ] and h ee-dimensional (3D) c ys als [ 13 ], bu ne e heless, he huge up u n in he ield was he in en ion o he ‘DNA o igami’ echnique [ 14 ] (Figu e 2a). The o igami app oach is based on olding a long single-s anded DNA sca old s and in o a desi ed shape wi h he help o a se o sho oligonucleo ides (s aples), and i has now become a widely accessible and exploi ed me hod o ab ica e cus om, modula and spa ially-well-de ined 2D [ 14 ] and 3D nanos uc u es wi h complex cu a u es, bends and wis s [15–18] (see Figu e 2b,c). La e on, me hods based on sca old- ee ab ica ion [ 19 ] (Figu e 2d), polyhed al ende ing [ 20 , 21 ] (Figu e 2e) and shape-complemen a i y [ 22 ] (Figu e 2 ) we e in oduced. Nanoma e ials 2016,6, 139 3 o 16 Nanoma e ials 2016, 6, 139 3 o 16 Figu e 2. (a) A DNA o igami echnique. A long sca old s and is olded in o a desi ed shape wi h he help o sho s aple s ands [14]; (b) Mul ilaye DNA o igami in squa e and honeycomb la ice [15,16]; (c) DNA o igami wi h cu a u es and bends [17,18]; (d) Sca old- ee ab ica ion o DNA nanoshapes. Nume ous a ge shapes can be ab ica ed by selec ing subse s o s ands om he cubic-like ‘molecula can as’ [19]; (e) A ully au oma ed op-down design me hod o c ea e meshed DNA o igami s uc u es [21]; ( ) DNA o igami s uc u es can be glued oge he by aking ad an age o he blun -end s acking and he shape-complemen a i y o he o igami uni s [22]. (a) is ep oduced wi h pe mission om [14]. Copy igh Na u e Publishing G oup, 2006. (b) is ep oduced wi h pe mission om [16]. Copy igh Na u e Publishing G oup, 2011. A sphe e in (c) is ep oduced wi h pe mission om [17]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2011. A gea -like objec in (c) is ep oduced wi h pe mission om [18]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2009. (d) is ep oduced wi h pe mission om [19]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2012. (e) is ep oduced wi h pe mission om [21]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2016. ( ) is ep oduced wi h pe mission om [22]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2015. In gene al, he DNA-based assembly o nanos uc u es is a highly pa allel echnique, and he nanome e -scale add essabili y o he c ea ed objec s makes i an in iguing app oach o de eloping no el bionano echnological applica ions [11]. To da e, loads o implemen a ions based on DNA nanos uc u es ha e been p esen ed, such as unable plasmonic de ices and me allic nanoshapes [23,24], ule s o op ical imaging [25], s uc u es o nanoelec onics [26,27], a i icial ion channels o anspo ing o sequencing molecules [28], and nano obo s o a ge ed d ug deli e y [29]. Mo eo e , as discussed in his e iew, DNA nanos uc u es p o ide an excellen ounda ion o designing enzyma ic eac o s and complex ca aly ic sys ems a he nanoscale. 2.2. DNA-Enzyme Conjuga es and A ays As discussed abo e, DNA s uc u es can be used as empla es o a ious molecules, ino ganic nanopa icles, and equally o unc ional enzymes [30]. Enzymes can be conjuga ed di ec ly o an oligonucleo ide (pa o a DNA s uc u e) o hey can be a ached o DNA h ough a speci ic binding mo i [30]. In gene al, i is impo an ha he enzyme ac i i y is e ained in he conjuga ion; a chosen enzyme should no be modi ied chemically o gene ically [31]. Fo example, sequence-speci ic DNA- binding p o eins can be used as adap o s in a achmen [32], and hei use can help o main ain he enzyme ac i i y in he conjuga ion. To da e, he e exis nume ous epo s o u ilizing simple nucleic acid mo i s o assemble unc ional enzymes and o o ganize chemical eac ions wi h p og ammabili y [33–36]. In addi ion, i has been shown ha by u ilizing DNA-based sel -assembly, s uc u ally-well-de ined p o ein a ays [30,37] and DNA-enzyme c ys als [7] can be c ea ed. Along hese lines, his e iew discusses ecen p og ess in c ea ing sma enzyme eac o s, dynamic egula o s, p o ein con aine s and ca ie s by aking ad an age o s a e-o - he-a DNA nanos uc u es, such as DNA o igami. 3. Enzyme Reac o s and Cascades Figu e 2. ( a ) A DNA o igami echnique. A long sca old s and is olded in o a desi ed shape wi h he help o sho s aple s ands [ 14 ]; ( b ) Mul ilaye DNA o igami in squa e and honeycomb la ice [ 15 , 16 ]; ( c ) DNA o igami wi h cu a u es and bends [ 17 , 18 ]; ( d ) Sca old- ee ab ica ion o DNA nanoshapes. Nume ous a ge shapes can be ab ica ed by selec ing subse s o s ands om he cubic-like ‘molecula can as’ [ 19 ]; ( e ) A ully au oma ed op-down design me hod o c ea e meshed DNA o igami s uc u es [ 21 ]; ( ) DNA o igami s uc u es can be glued oge he by aking ad an age o he blun -end s acking and he shape-complemen a i y o he o igami uni s [ 22 ]. ( a ) is ep oduced wi h pe mission om [ 14 ]. Copy igh Na u e Publishing G oup, 2006. ( b ) is ep oduced wi h pe mission om [ 16 ]. Copy igh Na u e Publishing G oup, 2011. A sphe e in ( c ) is ep oduced wi h pe mission om [ 17 ]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2011. A gea -like objec in ( c ) is ep oduced wi h pe mission om [ 18 ]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2009. ( d ) is ep oduced wi h pe mission om [ 19 ]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2012. ( e ) is ep oduced wi h pe mission om [ 21 ]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2016. ( ) is ep oduced wi h pe mission om [22]. Copy igh The Ame ican Associa ion o he Ad ancemen o Science, 2015. In gene al, he DNA-based assembly o nanos uc u es is a highly pa allel echnique, and he nanome e -scale add essabili y o he c ea ed objec s makes i an in iguing app oach o de eloping no el bionano echnological applica ions [ 11 ]. To da e, loads o implemen a ions based on DNA nanos uc u es ha e been p esen ed, such as unable plasmonic de ices and me allic nanoshapes [23,24] , ule s o op ical imaging [ 25 ], s uc u es o nanoelec onics [ 26 , 27 ], a i icial ion channels o anspo ing o sequencing molecules [ 28 ], and nano obo s o a ge ed d ug deli e y [ 29 ]. Mo eo e , as discussed in his e iew, DNA nanos uc u es p o ide an excellen ounda ion o designing enzyma ic eac o s and complex ca aly ic sys ems a he nanoscale. 2.2. DNA-Enzyme Conjuga es and A ays As discussed abo e, DNA s uc u es can be used as empla es o a ious molecules, ino ganic nanopa icles, and equally o unc ional enzymes [ 30 ]. Enzymes can be conjuga ed di ec ly o an oligonucleo ide (pa o a DNA s uc u e) o hey can be a ached o DNA h ough a speci ic binding mo i [ 30 ]. In gene al, i is impo an ha he enzyme ac i i y is e ained in he conjuga ion; a chosen enzyme should no be modi ied chemically o gene ically [ 31 ]. Fo example, sequence-speci ic DNA-binding p o eins can be used as adap o s in a achmen [ 32 ], and hei use can help o main ain he enzyme ac i i y in he conjuga ion. To da e, he e exis nume ous epo s o u ilizing simple nucleic acid mo i s o assemble unc ional enzymes and o o ganize chemical eac ions wi h p og ammabili y [ 33 – 36 ]. In addi ion, i has been shown ha by u ilizing DNA-based sel -assembly, s uc u ally-well-de ined p o ein a ays [ 30 , 37 ] and DNA-enzyme c ys als [ 7 ] can be c ea ed. Along hese lines, his e iew discusses ecen p og ess in c ea ing sma enzyme eac o s, dynamic egula o s, p o ein con aine s and ca ie s by aking ad an age o s a e-o - he-a DNA nanos uc u es, such as DNA o igami. Nanoma e ials 2016,6, 139 4 o 16 3. Enzyme Reac o s and Cascades An enzyme eac o ypically con ains one o mo e enzymes, which ca alyze a desi ed eac ion. The pu pose o he enzyme eac o is usually o maximize he eac ion e iciency ia compa men aliza ion o by b inging he eac ion coun e pa s in close p oximi y o each o he . By u ilizing designed DNA nanos uc u es wi h high add essabili y, enzymes can be a ached o hem wi h nanoscale p ecision. This is a key ac o o enzyme unc ions; a subs a e can only bind o an enzyme in a speci ic o ien a ion, and on he o he hand, he p oximi y o he compounds p o ided by he DNA empla es could signi ican ly enhance he enzyma ic eac ion a es [ 38 , 39 ]. In addi ion, i is essen ial o con ol he channeling o he subs a e and he eac ion in e media es o he enzyme cascades [ 40 ]. In many cases, compa men aliza ion could be used o e icien ly sepa a e and a ange simul aneous eac ions and eac ion compounds simila o complex na u al sys ems [ 41 ]. Mo eo e , enzyme eac o s can be equally u ilized o s udy enzyme unc ions and eac ion pa hways [ 42 ]. In his sec ion, ecen examples o using DNA nanos uc u es o build (s a ic) nano eac o s o biosensing and molecula -scale diagnos ics a e discussed (see also Table 1). Table 1. Examples o DNA-based enzyme eac o s and cascades. Type Func ion Key Aspec s A glucose oxidase (GOx) – ho se adish pe oxidase (HRP) cascade on a DNA o igami [43]. The enzyme posi ions on he DNA o igami empla e can be uned. The cascade ac i i y is highly dependen on he spacing be ween he enzymes; he highes ac i i y was ound a a 10 nm dis ance. A GOx-HRP cascade on a DNA o igami ha can be olled in o ubula shape [44]. The idea is simila o he abo e, bu he e he semi-con ined ubula geome y could enable shielding. The enzymes in he semi-con ined geome y show highe enzyma ic ac i i y han he ee enzyme con ols. A swinging a m be ween mala e dehyd ogenase (MDH) and glucose-6-phospha e dehyd ogenase (G6pDH) assembled on a double-c osso e (DX) DNA ile [45]. The DNA s and ac s as a lexible a m ha channels he co ac o ans e be ween he hyd ogenases in he complex. The enzyme ac i i y achie ed by he swinging a m is signi ican ly highe han in he case o eely di using co ac o . A ubula DNA o igami nano eac o wi h GOx-HRP pai s [46]. The nano eac o is comp ised o wo uni s: GOx- and HRP-loaded DNA o igamis ha can be combined in o a comple e cascade eac o . Single o igami uni s and he comple e eac o equipped wi h binding si es show highe ac i i y han he con ols wi hou binding si es. A xylose educ ase (XR) – xyli ol dehyd ogenase (XDR) cascade on a DNA o igami [47]. The enzymes a e a ached o o igami ia DNA-binding p o ein adap o s esul ing in an a i icial enzyme cascade. The e iciency o he cascade eac ion is mo e dependen on he in e enzyme dis ance han ha o he cascade eac ion wi h unimolecula anspo be ween wo enzymes. A h ee-enzyme pa hway assembled by a DNA nanos uc u e [48]. MDH, oxaloace a e deca boxylase (OAD) and lac a e dehyd ogenase (LDH) a e o ganized a he co ne s o he iangula DNA nanos uc u e, hus o ming a h ee-enzyme cascade. Ac i i y o he cascade depends mo e on he geome ic pa e ns o enzymes han he in e enzyme spacings. In he enzyme cascade sys em p esen ed in Figu e 3a, glucose oxidase (GOx) ca alyzes he oxidiza ion o glucose (subs a e) in he p esence o oxygen o gene a e gluconic acid and a hyd ogen pe oxide (H2O2) in e media e, which, in u n, se es as a subs a e o ho se adish pe oxidase (HRP) (HRP educes H 2 O 2 in o wa e ). Simul aneously, he p esence o H 2 O 2 esul s in he p o ona ion o he ABTS 2´ (2,2 1 -azinobis-(3-e hylbenz hiazoline-6-sul ona e) dianion, and hence, an ABTS ´ adical anion is gene a ed (ABTS ´ ac s as a epo e o he enzyme ac i i y). The di usion dis ance o he hyd ogen pe oxide limi s he a e o his enzyme cascade eac ion since HRP has a much highe u no e a e han GOx. Fu e al. s udied in e enzyme subs a e di usion by using a ec angula DNA o igami ile as a pla o m o p eo ganize GOx-HRP pai s in a dis ance-dependen manne [ 43 ]. The highes cascade ac i i y was ob ained when he in e enzyme dis ance was 10 nm. Impo an ly, he ac i i y was abou 15 imes highe han he con ol sample ha con ained unbound enzymes. A d as ic dec ease in ac i i y was obse ed as he in e enzyme dis ance was adjus ed o 20 nm, and he ac i i y was u he dec eased g adually as he dis ance was inc eased up o 65 nm. Nanoma e ials 2016,6, 139 5 o 16 Nanoma e ials 2016, 6, 139 5 o 16 anion is gene a ed (ABTS− ac s as a epo e o he enzyme ac i i y). The di usion dis ance o he hyd ogen pe oxide limi s he a e o his enzyme cascade eac ion since HRP has a much highe u no e a e han GOx. Fu e al. s udied in e enzyme subs a e di usion by using a ec angula DNA o igami ile as a pla o m o p eo ganize GOx-HRP pai s in a dis ance-dependen manne [43]. The highes cascade ac i i y was ob ained when he in e enzyme dis ance was 10 nm. Impo an ly, he ac i i y was abou 15 imes highe han he con ol sample ha con ained unbound enzymes. A d as ic dec ease in ac i i y was obse ed as he in e enzyme dis ance was adjus ed o 20 nm, and he ac i i y was u he dec eased g adually as he dis ance was inc eased up o 65 nm. Figu e 3. (a) A glucose oxidase (GOx) – ho se adish pe oxidase (HRP) enzyme cascade pai assembled on a ec angula DNA o igami [43]; (b) A ec angula DNA o igami shape wi h a ached enzyme cascade pai s (GOx and HRP) can be olled in o ubula shapes [44]; (c) A swinging a m o co ac o ans e be ween he enzymes (mala e dehyd ogenase (MDH) and glucose-6-phospha e dehyd ogenase (G6pDH)) assembled on a DNA ile [45]; (d) A modula and ubula DNA o igami- based enzyme cascade (GOx and HRP) nano eac o [46]; (e) An a i ical enzyme cascade (xylose educ ase (XR) and xyli ol dehyd ogenase (XDR)) pe o ming a co ac o coupled cascade eac ion on DNA o igami [47]; ( ) An a i icial h ee-enzyme (lac a e dehyd ogenase (LDH), MDH and oxaloace a e deca boxylase (OAD)) pa hway o ganized using a DNA nanos uc u e [48]. (a) is ep oduced wi h pe mission om [43]. Copy igh Ame ican Chemical Socie y, 2012. (b) is ep oduced wi h pe mission om [44]. Copy igh Ame ican Chemical Socie y, 2013. (c) is ep oduced wi h pe mission om [45]. Copy igh Na u e Publishing G oup, 2014. (d) is ep oduced wi h pe mission om [46]. Published by The Royal Socie y o Chemis y, 2015. (e) is ep oduced wi h pe mission om [47]. Copy igh Ame ican Chemical Socie y, 2016. ( ) is ep oduced wi h pe mission om [48]. Copy igh John Wiley and Sons, 2016. Inspi ed by he abo e-men ioned wo k, Fu e al. [44] designed ec angula (100 nm × 70 nm) DNA iles wi h GOx-HRP cascade pai s p ecisely posi ioned 15 nm apa om each o he . By using s icky-end ex ensions on he op and bo om edges o he DNA o igami ec angles, hey induced he ile o o m sho DNA nano ubes (Figu e 3b). E iciency o he enzyme cascade eac ion was quan i a i ely measu ed using an excess amoun o eac an glucose and he ch omogenic eac ion o he epo e ABTS2− (subs a e o HRP). The ac i i y was highes when he enzymes we e loca ed in a con ined nanospace wi hin he DNA nano ube. When he enzymes we e a ached o he semicon ined plana DNA ile, he ac i i y was lowe , bu s ill highe han ha o ee cascade con ols, which showed he lowes ac i i y. Hence, hese nanoscale bio eac o s p o ide access o an a i icial sys em o s udying biological p ocesses in o ganized cell-mimicking en i onmen s. Swinging a ms a e key cons i uen s o sequenced ca aly ic ans o ma ions in many na u ally occu ing mul i-enzyme complexes. The a m is commonly a chemical g oup co alen ly a ached o he Figu e 3. ( a ) A glucose oxidase (GOx) – ho se adish pe oxidase (HRP) enzyme cascade pai assembled on a ec angula DNA o igami [ 43 ]; ( b ) A ec angula DNA o igami shape wi h a ached enzyme cascade pai s (GOx and HRP) can be olled in o ubula shapes [ 44 ]; ( c ) A swinging a m o co ac o ans e be ween he enzymes (mala e dehyd ogenase (MDH) and glucose-6-phospha e dehyd ogenase (G6pDH)) assembled on a DNA ile [ 45 ]; ( d ) A modula and ubula DNA o igami-based enzyme cascade (GOx and HRP) nano eac o [ 46 ]; ( e ) An a i ical enzyme cascade (xylose educ ase (XR) and xyli ol dehyd ogenase (XDR)) pe o ming a co ac o coupled cascade eac ion on DNA o igami [ 47 ]; ( ) An a i icial h ee-enzyme (lac a e dehyd ogenase (LDH), MDH and oxaloace a e deca boxylase (OAD)) pa hway o ganized using a DNA nanos uc u e [ 48 ]. ( a ) is ep oduced wi h pe mission om [ 43 ]. Copy igh Ame ican Chemical Socie y, 2012. ( b ) is ep oduced wi h pe mission om [ 44 ]. Copy igh Ame ican Chemical Socie y, 2013. ( c ) is ep oduced wi h pe mission om [ 45 ]. Copy igh Na u e Publishing G oup, 2014. ( d ) is ep oduced wi h pe mission om [ 46 ]. Published by The Royal Socie y o Chemis y, 2015. ( e ) is ep oduced wi h pe mission om [ 47 ]. Copy igh Ame ican Chemical Socie y, 2016. ( ) is ep oduced wi h pe mission om [ 48 ]. Copy igh John Wiley and Sons, 2016. Inspi ed by he abo e-men ioned wo k, Fu e al. [ 44 ] designed ec angula (100 nm ˆ 70 nm) DNA iles wi h GOx-HRP cascade pai s p ecisely posi ioned 15 nm apa om each o he . By using s icky-end ex ensions on he op and bo om edges o he DNA o igami ec angles, hey induced he ile o o m sho DNA nano ubes (Figu e 3b). E iciency o he enzyme cascade eac ion was quan i a i ely measu ed using an excess amoun o eac an glucose and he ch omogenic eac ion o he epo e ABTS 2´ (subs a e o HRP). The ac i i y was highes when he enzymes we e loca ed in a con ined nanospace wi hin he DNA nano ube. When he enzymes we e a ached o he semicon ined plana DNA ile, he ac i i y was lowe , bu s ill highe han ha o ee cascade con ols, which showed he lowes ac i i y. Hence, hese nanoscale bio eac o s p o ide access o an a i icial sys em o s udying biological p ocesses in o ganized cell-mimicking en i onmen s. Swinging a ms a e key cons i uen s o sequenced ca aly ic ans o ma ions in many na u ally occu ing mul i-enzyme complexes. The a m is commonly a chemical g oup co alen ly a ached o he enzyme complex ia a lexible linke ha enables he di ec ans e o subs a e molecules be ween mul iple ac i e si es wi hin he complex. Fu e al. [ 45 ] cons uc ed a DNA nanos uc u e o assembling a mul i-enzyme sys em ha is equipped wi h an a i icial swinging a m. The a m was designed o e icien ly channel hyd ide ans e be ween wo dehyd ogenases. The whole design is illus a ed in Figu e 3c. The nanos uc u e complex u ilized a wo-enzyme cascade composed o glucose-6-phospha e dehyd ogenase (G6pDH) and malic dehyd ogenase (MDH) posi ioned on a DNA double-c osso e (DX) ile sca old. In he cascade sequence G6pDH ca alyzes he oxida ion Nanoma e ials 2016,6, 139 6 o 16 o glucose-6-phospha e and he educ ion o NAD + (nico inamide adenine dinucleo ide, oxidized) o NADH (nico inamide adenine dinucleo ide, educed). In he second cycle, MDH ca alyzes he educ ion o oxaloace a e o malic acid using he NADH p oduced by G6pDH. The swinging a m, an NAD + -equipped poly- hymine (poly-T) oligonucleo ide (20 nucleo ides long), was adhe ed o he DNA ile su ace exac ly hal way be ween he ancho ed enzymes G6pDH and MDH. The swinging a m’s capabili y o boos dehyd ogenase ac i i y in complexes con aining one enzyme coupled o a single NAD + a m was measu ed indi idually in bulk solu ion o h ee dis ances (7, 14 and 21 nm). The highes ac i i y o bo h G6pDH and MDH was obse ed a he 7 nm dis ance showing ca. 25- old enhancemen o ac i i y compa ed o an enzyme sys em in he p esence o he same concen a ion (100 nM) o eely di using NAD + . In he same expe imen al condi ions, he ac i i y o he ully assembled G6pDH–NAD + –MDH wo-enzyme nanos uc u e wi h a swinging a m is ca. 90- old highe han ha ob ained using he same wo-enzyme complex bu wi h eely di using NAD+. Linko e al. [ 46 ] designed and ab ica ed an enzyme eac o , which consis s o wo dis inc ubula 3D DNA o igami building blocks wi h ei he GOx o HRP enzymes ancho ed inside he o igami compa men h ough bio in–Neu A idin (NTV) binding (Figu e 3d). Bo h uni s we e ab ica ed sepa a ely, and ‘glued’ oge he ia a p og ammable DNA base-pai ing by hyb idizing 32 sho ( h ee o six bases) sequences. The sho sequences ha we e s icking ou a he end o one uni we e pai ed wi h ee sca old si es loca ed a he edge o ano he uni . The o he end o he o igami uni was passi a ed by o e hanging single-s anded poly-T sequences (8 nucleo ides) in o de o p e en he o ma ion o mul ime s. The ca aly ic ac i i y o a wo-uni nano eac o was moni o ed in he en i onmen con aining excess amoun s o D-glucose as a eac an and 3,3 1 ,5,5 1 - e ame hylbenzidine (TMB) as a epo e in o de o achie e a eac ion ha is es ic ed by he di usion a e o he in e media e p oduc H 2 O 2 . Compa ed o he con ol samples (simila ly p epa ed samples bu wi hou NTV binding si es o enzymes), he assembled win-uni nano eac o has much highe ac i i y, hus indica ing ha unspeci ic binding be ween enzymes and o igami s uc u es is insigni ican . Ngo e al. [ 47 ] in oduced co ac o -coupled cascade eac ions on a DNA o igami sca old. The cascade was based on he D-xylose me abolic pa hway, and combined wo enzymes: xylose educ ase (XR) and xyli ol dehyd ogenase (XDR). The enzymes we e a ached o he DNA sca old wi h DNA-binding p o ein adap o s, he zinc inge p o ein (zi 268) and he basic leucine-zippe p o ein (GCN4). The cascade mechanism elies on he ecycling o co ac o NADH be ween he enzymes, which is possible due o hei close p oximi y. Wi hin he me abolic pa hway o xylose, he i s enzyme XR con e s xylose in o xyli ol by consuming he co ac o NADH. The p oduced xyli ol and NAD + a e bo h simul aneously anspo ed o he second enzyme XDH, which con e s xyli ol in o xylulose by consuming NAD+ o ecycle he NADH co ac o (Figu e 3e). Liu e al. [ 48 ] assembled an a i icial h ee-enzyme pa hway on a se ies o DNA nanosca olds in o de o s udy he dependence o hei ac i i ies. They measu ed he ac i i ies o an MDH-OAD-LDH (mala e dehyd ogenase–oxaloace a e deca boxylase–lac a e dehyd ogenase) cascade wi h a iable spa ial dis ances and geome ic a angemen s. The h ee-enzyme pa hway (Figu e 3 ) s a s wi h he MDH-ca alyzed oxida ion o malic acid o oxaloace a e (OAA) and he simul aneous educ ion o NAD + o NADH. In he nex cycle OAD con e s OAA in o py u ic acid and inhibi s i s con e sion back o malic acid. In he hi d cycle LDH consumes he educed NADH and py u ic acid o p oduce lac ic acid. Unlike he abo e-men ioned wo-enzyme sys ems, he o e all ac i i y o he h ee-enzyme pa hway was mo e dependen on he geome ic pa e ns ha a anged enzymes wi hin a sho dis ance (10–30 nm) o each o he a he han wi h in e enzyme spacings. By op imizing he geome ic pa e ns o he h ee enzymes, a i e- old ac i i y enhancemen was ob ained compa ed o he unassembled ee enzymes. In addi ion, he deple ion o he pa hway in e media es was e y e icien in he assembled enzyme sys ems wi h li le de ec able NADH in he bulk solu ion, indica ing ha nea ly all NADH was coupled in o he enzyme pa hway wi hou leakage. Nanoma e ials 2016,6, 139 7 o 16 4. Enzyma ic Nanode ices wi h Mo ion Besides he s a ic nano eac o s discussed in he p e ious sec ion, he e a e compelling examples o in i o nanode ices ha can con ol enzyme ac i i y. These de ices can be swi ched be ween an ac i e and inac i e s a e by in oducing a speci ic igge . The igge s a e usually DNA s ands ha a e able o pe o m p ep og ammed s and displacemen eac ions. Al e na i ely, some o he sys ems can au onomously egula e he eac ion(s). He e, a ew examples o mechanical egula o y DNA-enzyme de ices, au onomous molecula sys ems and hei wo king p inciples a e e iewed (see also Table 2). Table 2. Examples o mechanical egula o y DNA-enzyme de ices. Type Func ion Key Aspec s DNA nano weeze s [49–52] equipped wi h cascade pai s o wi h he enzyme and i s co ac o . The weeze s can be opened and closed h ough a s and-displacemen eac ion. The enzyme ac i i y can be con olled by swi ching he weeze s e e sibly. A ubula DNA o igami nano eac o [53]. The lid o he ube can be opened and closed wi h he help o lock and key s ands. Flow h ough o he compounds in o he con ined eac ion chambe is con olled by he lid. A ou -a m DNA o igami nanoac ua o [54]. A dis ance change in a d i e si e can be p opaga ed o he mi o si e con aining binding si es o ca go molecules. The ac ua o can be d i en using di e en mechanisms, and i can be used o , e.g., uning luo escence beha io o enhanced luo escen p o ein (eGFP). Ap ame -based logical ci cui [ 55 ]. The au onomous logical ci cui con ols α- h ombin ac i i y h ough he con e o , con olle and gene a o modules. α- h ombin aids blood coagula ion, and he e o e sys ems such as his may ind in iguing biomedical uses. 4.1. Mechanical Regula o y DNA-Enzyme De ices Liu e al. [ 49 ] employed a DNA weeze nanos uc u e o ac ua e he eac ion be ween a G6pDH/NAD + enzyme-co ac o pai . In his cons uc (Figu e 4a), he enzyme and co ac o we e a ached o wo di e en ca. 14-nm-long a ms. Ac ua ion o he enzyme unc ion was achie ed by swi ching be ween open and closed s a es o he weeze s, in o he wo ds by spa ially sepa a ing he enzyme-co ac o pai o inhibi ion o b inging he pai oge he o ac i a ion, espec i ely. In he eac ion cycle, NAD + is i s educed o NADH by G6pDH. Then, phenazine me hosul a e (PMS) ca alyzes elec on ans e om NADH o esazu in, which p oduces s ongly luo escen eso u in. In he weeze geome y, a 25-nucleo ide (n ) ssDNA oligome connec ed he ends o he weeze a ms and se ed as a s uc u al egula o y elemen o con ol he s a e o he sys em. The open s a e can be a ained by dis up ing he hai pin ia hyb idiza ion be ween a complemen a y se s and and a hai pin, hus gene a ing a igid ca. 16-nm-long dsDNA domain be ween he ends o he weeze a ms. By adding a uel s and ( ully complemen a y o he se s and) o he sys em, a hai pin is eleased by a s and-displacemen mechanism and he weeze s a e swi ched back o he closed s a e. Opening and closing mechanisms ha e been u he op imized by Dhakal e al. [50]. Mo eo e , Xin e al. [ 51 ] used simila nano weeze s and chose he GOx-HRP cascade as a model o demons a e he e e sible egula ion o he enzyme cascade eac ion. The DNA machine was comp ised o double-c osso e (DX) mo i s, which o med wo igid a ms (glued oge he by an immobile ou -way junc ion). A DNA mo o , which can swi ch be ween a s em-loop and a double-helix s uc u e d i en by a s and displacemen eac ion, was inco po a ed in o he middle o he DNA machine o cycle be ween open and closed s a es. This kind o de ice could also be used o e e sibly egula e he a ge binding a ini y o a h ombin p o ein, as shown by Chou e al. [52]. Nanoma e ials 2016,6, 139 8 o 16 Nanoma e ials 2016, 6, 139 8 o 16 DNA machine o cycle be ween open and closed s a es. This kind o de ice could also be used o e e sibly egula e he a ge binding a ini y o a h ombin p o ein, as shown by Chou e al. [52]. Figu e 4. (a) Nano weeze s o egula e enzyme ac i i y [49]; (b) Tubula nano eac o wi h swi chable lid o con ol he low h ough o he eac ion compounds [53]; (c) DNA o igami nanoac ua o ha can be d i en by, e.g., single-s anded DNA (ssDNA) s ands o es ic ion enzymes [54]; (d) Ap ame - based logical molecula ci cui o con ol h ombin ac i i y [55]. (a) is ep oduced wi h pe mission om [49] Copy igh Na u e Publishing G oup, 2013. (b) is ep oduced wi h pe mission om [53]. Copy igh The Royal Socie y o Chemis y, 2016. (c) is ep oduced wi h pe mission om [54]. Published by Na u e Publishing G oup, 2016. (d) is ep oduced wi h pe mission om [55]. Copy igh Ame ican Chemical Socie y, 2012. Wang e al. [53] p epa ed a DNA o igami nanochannel as a sca old o moni o ing he GOx- HRP cascade eac ion. The channel, 100 nm in leng h and 22 nm in diame e , was o med by olling up a ec angula o igami objec wi h he help o s icky ends, placed as ex ensions a he op and bo om helices o he shee -like s uc u e (depic ed in Figu e 4b). A ow o 11 s aple s ands, called shu e s ands, which con ain 15 nucleo ides long o e hangs in an up igh posi ion o he conca e side, o med a shu e a he end o he nanochannel which can con ol he opening and closing o he channel upon s imuli. By adding he ‘lock s ands’, i.e., ssDNA molecules complemen a y o he 15 Figu e 4. ( a ) Nano weeze s o egula e enzyme ac i i y [ 49 ]; ( b ) Tubula nano eac o wi h swi chable lid o con ol he low h ough o he eac ion compounds [ 53 ]; ( c ) DNA o igami nanoac ua o ha can be d i en by, e.g., single-s anded DNA (ssDNA) s ands o es ic ion enzymes [ 54 ]; ( d ) Ap ame -based logical molecula ci cui o con ol h ombin ac i i y [ 55 ]. ( a ) is ep oduced wi h pe mission om [ 49 ] Copy igh Na u e Publishing G oup, 2013. ( b ) is ep oduced wi h pe mission om [ 53 ]. Copy igh The Royal Socie y o Chemis y, 2016. ( c ) is ep oduced wi h pe mission om [ 54 ]. Published by Na u e Publishing G oup, 2016. ( d ) is ep oduced wi h pe mission om [ 55 ]. Copy igh Ame ican Chemical Socie y, 2012. Wang e al. [ 53 ] p epa ed a DNA o igami nanochannel as a sca old o moni o ing he GOx-HRP cascade eac ion. The channel, 100 nm in leng h and 22 nm in diame e , was o med by olling up a ec angula o igami objec wi h he help o s icky ends, placed as ex ensions a he op and bo om helices o he shee -like s uc u e (depic ed in Figu e 4b). A ow o 11 s aple s ands, called shu e s ands, which con ain 15 nucleo ides long o e hangs in an up igh posi ion o he conca e side, o med a shu e a he end o he nanochannel which can con ol he opening and closing o he channel upon s imuli. By adding he ‘lock s ands’, i.e., ssDNA molecules complemen a y o he 15 n o e hangs, igid DNA duplexes we e o med, esul ing in an e icien closing o he shu e . 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